High Molecular Weight Isoforms Research Articles

Serum CTRP9 and high-molecular weight adiponectin are associated with ischemic stroke

BackgroundC1q/TNF-related protein 9 (CTRP9) and adiponectin (APN) have beneficial metabolic regulatory and vasoprotective effects. This study explored alteration of CTRP9 and APN multimers during onset of ischemic stroke and development, to provide novel clinical and experimental basis for recognition and prevention of ischemic stroke.MethodsThere were 269 patients with ischemic stroke and 182 control subjects included in this study. Serum levels of CTRP9 and APN multimers in different disease stages were measured.ResultsSerum CTRP9, total APN (tAPN), and high-molecular weight (HMW) APN decreased gradually in stage I (acute stage, within 72 h of onset) of ischemic stroke and increased during stage III (11th day to one month) and stage IV (1 month after), compared to control. In the non-hyperlipidemia group, serum CTRP9, tAPN, and HMW were decreased in ischemic stroke patients compared to control (P < 0.05). Serum CTRP9 is closely related to serum tAPN and HMW (r = 0.992, 0.991). Serum CTRP9 are protective against ischemic stroke (OR = 0.400, 95% CI 0.197–0.810, P < 0.05).ConclusionsLower serum CTRP9, tAPN, LMW, and HMW are significantly associated with increased ischemic stroke risk in non-hyperlipidemia subjects. CTRP9, tAPN, and HMW isoforms may be valuable clinical indicators for patients with ischemic stroke.

PSUN311 JTV-519 Alleviates the Impairment of FGF21 Axis Signaling after Spinal Cord Contusion in Mice

Abstract Spinal cord injury (SCI) causes adverse changes in body composition and metabolic profile due to dramatic loss of skeletal muscle mass and an extremely sedentary lifestyle. These abrupt and permanent changes increase the risk for cardiovascular disease, liver disorders, and type 2 diabetes compared to the general population. We have reported that metabolic dysregulation following SCI due to transection is associated with impaired fibroblast growth factor 21 (FGF21) and adiponectin (ADPN) signaling [1], two important regulators of metabolism [2, 3]. JTV519, which stabilizes ryanodine receptors and blocks calcium uptake via SERCA [4], has been reported to have a strong cardioprotective effect via stabilization of ryanodine receptor 2. We examined the effects of SCI caused by contusion (a more common cause of SCI), with or without JTV519 administration, on FGF21 and ADPN signaling in tissues collected from mice at 56 days after SCI. SCI reduced serum and hepatic mRNA levels of FGF21 by 75% and 45%, respectively, compared to sham mice. SCI also significantly diminished FGF21 receptor (FGF-R1) and coreceptor (beta-klotho, KLB) mRNA expression in adipose tissue. In addition, SCI decreased serum ADPN levels, particularly its high molecular weight (HMW) isoform which is the most potent form of the protein. These inhibitory effects were associated with decreased hepatic mRNA expression for PPARalpha (55%), type-2 adiponectin receptor (AdipoR2; 70%), and Glut4 (75%). In addition, SCI reduced mRNA expression of leptin (by 85%) and UCP1 (by 95%) in adipose tissue. In skeletal muscle (gastrocnemius), SCI lowered mRNA expression of PGC1alpha (by 38%), ADPN (by 80%), FGF-R1 (by 35%) and AdipoR1 (by 20%) compared to sham mice. Treating sham-mice with JTV519 upregulated FGF21 and ADPN serum and mRNA levels as well as the expression of the associated metabolic genes of interest (mentioned above). Likewise, SCI-mice treated with JTV519 also demonstrated upregulation of serum levels of FGF21, the HMW isoform of ADPN, and hepatic FGF21 mRNA expression compared to vehicle-treated SCI-mice. Decreased hepatic mRNA expression of PPARalpha, AdipoR2, Glut4 and FGF-R1 and KLB mRNA in adipose tissue were also reversed after JTV519 administration, with levels approximately equivalent to those observed in sham-vehicle treated mice but lower than sham-JTV519 treated mice. Moreover, JTV519 treatment protected against SCI-induced decreases of PGC1alpha, ADPN and FGF-R1 mRNA expression in the gastrocnemius. These data imply that JTV-519 is able to alleviate SCI-induced impairments of FGF21/ADPN signaling. Therefore, JTV519 could be a potential pharmacological agent to treat metabolic dysfunction after SCI.

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

Elimination of endogenous high molecular weight FGF2 prevents pressure-overload-induced systolic dysfunction, linked to increased FGFR1 activity and NR1D1 expression.

Fibroblast growth factor 2 (FGF2), produced as high (Hi-) and low (Lo-) molecular weight isoforms, is implicated in cardiac response to injury. The role of endogenous FGF2 isoforms during chronic stress is not well defined. We investigated the effects of endogenous Hi-FGF2 in a mouse model of simulated pressure-overload stress achieved by transverse aortic constriction (TAC) surgery. Hi-FGF2 knockout mice, expressing only Lo-FGF2, FGF2(Lo), and wild-type mice, FGF2(WT), expressing both Hi-FGF2 and Lo-FGF2, were used. By echocardiography, a decline in systolic function was observed in FGF2(WT) but not FGF2(Lo) mice compared to corresponding sham-operated animals at 4-8weeks post-TAC surgery. TAC surgery increased markers of myocardial stress/damage including B-type natriuretic peptide (BNP) and the pro-cell death protein BCL2/adenovirus E1B 19kDa protein-interacting protein-3 (Bnip3) in FGF2(WT) but not FGF2(Lo) mice. In FGF2(Lo) mice, cardiac levels of activated FGF receptor 1 (FGFR1), and downstream signals, including phosphorylated mTOR and p70S6 kinase, were elevated post-TAC. Finally, NR1D1 (nuclear receptor subfamily 1 group D member 1), implicated in cardioprotection from pressure-overload stress, was downregulated or upregulated in the presence or absence, respectively, of Hi-FGF2 expression, post-TAC surgery. In wild-type cardiomyocyte cultures, endothelin-1 (added to simulate pressure-overload signals) caused NR1D1 downregulation and BNP upregulation, similar to the effect of TAC surgery on the FGF2(WT) mice. The NR1D1 agonist SR9009 prevented BNP upregulation, simulating post-TAC findings in FGF2(Lo) mice. We propose that elimination of Hi-FGF2 is cardioprotective during pressure-overload by increasing FGFR1-associated signaling and NR1D1 expression.

Review on Adiponectin: A Benevolent Adipokine

Adiponectin is a most abundant secretory protein produced by adipocytes of white adipose tissue. Adiponectin circulates in blood as three different (high-molecular, middle-molecular, and low-molecular weight) isoforms, gives its effects through AdipoR1 and AdipoR2 receptor. Primary data suggesting that adiponectin has insulin-sensitizing, anti-atherogenic, and anti-inflammatory effects. High serum level of adiponectin is positively associated with inflammation severity and pathological progression in chronic kidney disease, liver disease and inflammatory bowel disease. It has emerged as a valuable biomarker for insulin sensitivity, cardiovascular risk and inflammation. Adiponectin is gaining attention for its therapeutic role in Alzheimer’s disease. Adiponectin appears to play a crucial role not only in glucose and lipid metabolism but also the development and progression of different cancers. Adiponectin also produced locally in the retinas participate in defense of various eye diseases. This review summarizes the role of adiponectin as benevolent adipokine in different disorders.

What Can We Learn from FGF-2 Isoform-Specific Mouse Mutants? Differential Insights into FGF-2 Physiology In Vivo.

Fibroblast growth factor 2 (FGF-2), ubiquitously expressed in humans and mice, is functionally involved in cell growth, migration and maturation in vitro and in vivo. Based on the same mRNA, an 18-kilo Dalton (kDa) FGF-2 isoform named FGF-2 low molecular weight (FGF-2LMW) isoform is translated in humans and rodents. Additionally, two larger isoforms weighing 21 and 22 kDa also exist, summarized as the FGF-2 high molecular weight (FGF-2HMW) isoform. Meanwhile, the human FGF-2HMW comprises a 22, 23, 24 and 34 kDa protein. Independent studies verified a specific intracellular localization, mode of action and tissue-specific spatiotemporal expression of the FGF-2 isoforms, increasing the complexity of their physiological and pathophysiological roles. In order to analyze their spectrum of effects, FGF-2LMW knock out (ko) and FGF-2HMWko mice have been generated, as well as mice specifically overexpressing either FGF-2LMW or FGF-2HMW. So far, the development and functionality of the cardiovascular system, bone formation and regeneration as well as their impact on the central nervous system including disease models of neurodegeneration, have been examined. This review provides a summary of the studies characterizing the in vivo effects modulated by the FGF-2 isoforms and, thus, offers a comprehensive overview of its actions in the aforementioned organ systems.

The effects of blanching on composition and modification of proteins in navy beans (Phaseolus vulgaris)

Blanching is an important process in the preparation of navy beans (Phaseolus vulgaris L.) for canning. We here explore the effect of blanching which can profoundly affect protein composition and introduce protein-primary-level modifications. Amino acid analysis showed significantly decreased protein abundance (58.5%) in blanched beans compared to raw beans. Proteomic analyses revealed a decrease in high molecular weight isoforms of the major storage globulin proteins phaseolin (mean fold-change −3.7) and legumin (mean fold-change −2.5) and concomitant increase in their low molecular weight isoforms (mean fold-change 6.4 and 8.3, respectively). Blanched beans also had decreased abundance of lipoxygenase (mean fold-change −13.1), an enzyme responsible for product spoilage during storage. Increased lysinoalanine (up to 47%) and highly modified protein fragments were found in the processing waters, indicating heat- induced modifications. Correlating these molecular level changes thus provides a basis for evaluating how processing parameters can be modified to increase protein food quality.

P.488 Effects of acute and chronic administration of a delta opioid receptor ligands on the excitability of serotonin neurons in rats

Fibroblast growth factor-2 (FGF-2) is a potent neurotrophic factor promoting survival of dopaminergic (DA) neurons in vitro and in vivo. FGF-2 is expressed in different isoforms representing distinct translation products from a single mRNA. For this study, we focused on the high molecular weight (HMW) isoform, which, after non-viral plasmid-based overexpression in embryonic day 12 (E12) rat ventral mesencephalon (VM)-derived cells, revealed increased numbers of tyrosine hydroxylase-positive (TH+) cells in a ‘colayer’ cell culture model. To determine the therapeutic potential of VM cells producing FGF-2-HMW as their ‘own’ neurotrophic factor, we transplanted cell suspensions obtained from such in vitro modified and differentiated cell cultures into the 6-hydroxydopamine (6-OHDA) hemiparkinsonian rat model. Animals, having received either non-transfected cells, empty-control transfected, or FGF-2-HMW-plasmid transfected cells, were analyzed in two different transplantation paradigms each using 172,000 or 520,000 cells, respectively. The behavioral performances in the amphetamine- and apomorphine-induced rotational test as well as in the cylinder test were evaluated for up to thirteen weeks post transplantation (postTX). Finally, the integration of the grafted cells into the host striatum was analyzed by immunohistochemical measurements. Those analyses revealed improvements of behavioral deficits in all five groups receiving DA neuron grafts, except for amphetamine-induced rotation of the FGF-2-HMW small graft group. Altogether, genetic modification with the FGF-2-HMW-plasmid did not further improve functional recovery compared to the control groups and had no influence on either the number of surviving DA neurons or on the density of outgrowing TH+ fibers.

Increased high molecular weight adiponectin and lean mass during tocilizumab treatment in patients with rheumatoid arthritis: a 12-month multicentre study

BackgroundPatients with rheumatoid arthritis (RA) have an increased risk of cardiovascular (CV) disease. Adiponectin is involved in the metabolism of glucose and lipids with favourable effects on CV disease, especially its high molecular weight (HMW) isoform. Body composition changes are described in RA with various phenotypes including obesity. The effects of tocilizumab on serum adiponectin and body composition, especially fat mass, in patients with RA are not well determined.MethodsPatients with active RA despite previous csDMARDs and/or bDMARDs and who were tocilizumab naïve were enrolled in a multicentre open-label study. They were evaluated at baseline, 1, 3, 6 and 12 months. Clinical assessment included body mass index (BMI) and anthropometric measurements. Lipid and metabolic parameters, serum adiponectin (total and HMW), leptin, resistin and ghrelin were measured at each time point. Body composition (lean mass, fat mass, % fat, fat in the android and gynoid regions) was evaluated at baseline, 6 and 12 months.ResultsOne hundred seven patients were included. Both total and HMW adiponectin significantly increased from baseline to month 3, peaking respectively at month 3 (p = 0.0105) and month 1 (p < 0.0001), then declining progressively until month 6 to 12 and returning to baseline values. Significant elevation in HMW adiponectin persisted at month 6 (p = 0.001). BMI and waist circumference significantly increased at month 6 and 12, as well as lean mass at month 6 (p = 0.0097). Fat mass, percentage fat and android fat did not change over the study period. Lipid parameters (total cholesterol and LDL cholesterol) increased while glycaemia, insulin and HOMA-IR remained stable. Serum leptin, resistin and ghrelin did not change during follow-up.ConclusionsTocilizumab treatment in RA patients was associated with a significant increase in total and HMW adiponectin, especially at the onset of the treatment. Tocilizumab also induced a significant gain in lean mass, while fat mass did not change. These variations in adiponectin levels during tocilizumab treatment could have positive effects on the CV risk of RA patients. In addition, tocilizumab may have an anabolic impact on lean mass/skeletal muscle.Trial registrationThe ADIPRAT study was a phase IV open-label multicentre study retrospectively registered on ClinicalTrials.gov under the number NCT02843789 (date of registration: July 26, 2016).

Phosphoproteomic analysis identifies phospho-Threonine-17 site of phospholamban important in low molecular weight isoform of fibroblast growth factor 2-induced protection against post-ischemic cardiac dysfunction

RationaleAmong its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. ObjectiveYet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. Methods and resultsTo identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Selected Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05). ConclusionThe findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction.

Changes in gene expression caused by genetic elimination of high molecular weight FGF2 are associated with prevention of stress‐induced cardiac systolic dysfunction

Fibroblast growth factor 2 (FGF2) is produced as high (&gt;20 kDa, Hi‐) and low molecular weight (18 kDa, Lo) isoforms in the heart. While administered Lo‐FGF2 has been established as a cardioprotective agent in multiple models of cardiac injury, there is limited information about the role of Hi‐FGF2 in the heart. To investigate the effect of endogenous Hi‐FGF2 we compared cardiac transcriptome (35240 targets, GeneChip™ Mouse Gene 2.0 ST Array (Affymetrix, 902119)) and systolic function between Hi‐FGF2 knock‐out mice, FGF2(Lo), expressing only Lo‐FGF2, and wild type mice, FGF2(WT), expressing both Hi‐FGF2 and Lo‐FGF2, under ‘normal’ (sham‐operated) and “stress” (pressure overload) conditions. Transaortic constriction surgery (TAC) was used to induce pressure overload. Echocardiography was done at baseline and at 4–8 weeks post‐surgery. Microarray analysis showed that under normal conditions, the absence of Hi‐FGF2 promoted changes in relative levels of 118 transcripts, including those associated with circadian rhythm regulation and heat shock protein (HSP70)‐associated apoptosis regulation. Comparative analysis of TAC surgery‐induced gene expression changes showed that 275 transcripts were differentially affected by the presence or absence of Hi‐FGF2 expression, most prominently the orphan nuclear receptor NR1D1 which is linked to regulation of circadian rhythm and metabolism. Under non‐stress conditions, systolic function was unaffected by endogenous Hi‐FGF2 expression. Pressure overload stress caused a decline in systolic function in the presence of endogenous Hi‐FGF2, at 4–8 weeks post‐TAC surgery, accompanied by increases in markers of myocardial stress/damage including B‐type natriuretic peptide (BNP) and the pro‐cell death protein BCL2/adenovirus E1B 19 kDa protein‐interacting protein‐3, Bnip3. In the absence of endogenous Hi‐FGF2, mice were protected from stress‐induced loss of systolic function and increases in BNP and Bnip3. TAC surgery induced Hi‐FGF2‐independent increases in: cardiac mass (heart weight/tibia length); cardiac fibrosis; transcripts linked to exracellular matrix remodeling. Increased cardiomyocyte size, however, was only observed in FGF2(WT) but not FGF2(Lo) hearts post‐TAC. It is suggested that elimination of endogenous Hi‐FGF2 elicits cardioprotection by increasing cardiac HSP70 (pre‐TAC surgery); and NR1D1, after TAC surgery.Support or Funding InformationFunding (EK, PAC, DJ) was provided by the Canadian Institutes for Health Research (FRN‐74733) and the Molson Women’s Heart Health Foundation (EK). MPC was supported by a CIHR Open Operating Grant (MOP136862). NK and RSN were the recipients of a Bank of Montreal studentship award via the St. Boniface Hospital Albrechtsen Research Centre and University of Manitoba funding to PAC.

Multifaceted Physiological Roles of Adiponectin in Inflammation and Diseases.

Adiponectin is the richest adipokine in human plasma, and it is mainly secreted from white adipose tissue. Adiponectin circulates in blood as high-molecular, middle-molecular, and low-molecular weight isoforms. Numerous studies have demonstrated its insulin-sensitizing, anti-atherogenic, and anti-inflammatory effects. Additionally, decreased serum levels of adiponectin is associated with chronic inflammation of metabolic disorders including Type 2 diabetes, obesity, and atherosclerosis. However, recent studies showed that adiponectin could have pro-inflammatory roles in patients with autoimmune diseases. In particular, its high serum level was positively associated with inflammation severity and pathological progression in rheumatoid arthritis, chronic kidney disease, and inflammatory bowel disease. Thus, adiponectin seems to have both pro-inflammatory and anti-inflammatory effects. This indirectly indicates that adiponectin has different physiological roles according to an isoform and effector tissue. Knowledge on the specific functions of isoforms would help develop potential anti-inflammatory therapeutics to target specific adiponectin isoforms against metabolic disorders and autoimmune diseases. This review summarizes the current roles of adiponectin in metabolic disorders and autoimmune diseases.

FGF-2 isoforms influence the development of dopaminergic neurons in the murine substantia nigra, but not anxiety-like behavior, stress susceptibility, or locomotor behavior

BackgroundLoss of fibroblast growth factor 2 (FGF-2) is responsible for the development of an increased number of dopaminergic (DA) neurons in the murine substantia nigra pars compacta (SNpc). Furthermore, dysregulation of its expression patterns within the central nervous system (CNS) is associated with behavioral abnormalities in mice. Until now, the contributions of the individual FGF-2 isoforms (one low (LMW) and two high molecular weight (HMW) isoforms) in the CNS are elusive. MethodsTo unravel the specific effects of FGF-2 isoforms, we compared three knockout mouse lines, one only deficient for LMW, one deficient for HMW and another lacking both isoforms, regarding DA neuronal development. With this regard, three time points of ontogenic development of the SNpc were stereologically investigated. Furthermore, behavioral aspects were analyzed in young adult mice, supplemented by corticosterone measurements. ResultsJuvenile mice lacking either LMW or HMW develop equal supernumerary DA neuron numbers in the SNpc. Compensatory increased LMW expression is observed in animals lacking HMW. Meanwhile, no knockout mouse line demonstrated changes in anxiety-like behavior, stress susceptibility, or locomotor behavior. ConclusionsBoth FGF-2 isoforms crucially influence DA neuronal development in the murine SNpc. However, absence of LMW or HMW alone alters neither anxiety-like nor locomotor behavior, or stress susceptibility. Therefore, FGF-2 is not a determinant and causative factor for behavioral alterations alone, but probably in combination with appropriate conditions, like environmental or genetic factors.

Abstract 838: High Molecular Weight FGF2 Contributes to Pressure Overload Induced Systolic Dysfunction by a Mechanism Associated With Modulation of the NR1D1 Orphan Nuclear Receptor Expression

Fibroblast growth factor 2 (FGF2) is implicated in normal cardiac development as well as cardiac pathophysiology; however, FGF2 exist as multiple high and low molecular weight isoforms. While endogenous low molecular weight FGF2 (Lo-FGF2) is cardioprotective during chronic stress, the more prevalent endogenous high molecular weight FGF2 (Hi-FGF2) is proposed to promote maladaptive cardiac remodeling. We have investigated the hypothesis that genetic elimination of Hi-FGF attenuates cardiac dysfunction in mice that have been subjected to pressure overload by transverse aortic constriction (TAC). Two groups of male C57BL/6mice were compared: (1) Wild type (WT) mice, expressing Hi- and Lo-FGF2 (FGF[WT] mice); and (2) Hi-FGF2 knock-out mice, expressing only Lo-FGF2 (FGF[Lo] mice). Echocardiographic assessment of heart function and dimensions was done at baseline and then 4 and 8 weeks after TAC or sham surgery. FGF[WT] mice displayed a decline in systolic function compared to their corresponding sham animals at 4- and 8-weeks post-TAC, which was absent in the FGF[Lo] mice. Relative levels of B-type natriuretic peptide, a marker of cardiac pathology severity, were elevated in FGF[WT] but not FGF[Lo] mice compared to shams. Increased accumulation of the pro-cell death protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 was more pronounced in the FGF(WT) compared to FGF(Lo) mice, post TAC. Microarray analysis of the whole transcriptome of hearts in FGF2[WT] and FGF2[Lo] mice indicated the pathway linked to circadian rhythm as a candidate for the most significant differentially regulated. Specifically, upregulation of the circadian rhythm master regulator, Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1), was validated by qPCR and protein immunoblotting in FGF[Lo] mice versus downregulation of NR1D1in FGF[WT] mice post-TAC, when compared to their sham operated littermates. Taken together these studies suggest that expression of Hi-FGF2 contributes to cardiac systolic dysfunction in left ventricular pressure overloaded WT mice by downregulation of Nr1D1, post-TAC.

Isoform-selective as opposed to complete depletion of fibroblast growth factor 2 (FGF-2) has no major impact on survival and gene expression in SOD1G93A amyotrophic lateral sclerosis mice.

We have previously shown that total knockout of fibroblast growth factor-2 (FGF-2) results in prolonged survival and improved motor performance in superoxide dismutase 1 (SOD1G93A ) mutant mice, the most widely used animal model of the fatal adult onset motor neuron disease amyotrophic lateral sclerosis (ALS). Moreover, we found differential expression of growth factors in SOD1G93A mice, with distinct regulation patterns of FGF-2 in spinal cord and muscle tissue. Within the present study we aimed to characterize FGF-2-isoform specific effects on survival, motor performance as well as gene expression patterns predominantly in muscle tissue by generating double mutant SOD1G93A FGF-2 high molecular weight- and SOD1G93A FGF-2 low molecular weight-knockout mice. While isoform specific depletion was not beneficial regarding survival or motor performance of double mutant mice, we found isoform-dependent differential gene expression of epidermal growth factor (EGF) in the muscle of SOD1G93A FGF-2 low molecular weight knockout mice compared to single mutant SOD1G93A mice. This significant downregulation of EGF in the muscle tissue of double mutant SOD1G93A FGF-2 low molecular weight knockout mice implies that FGF-2 low molecular weight knockout (or the presence of the FGF-2 high molecular weight isoform) selectively impacts EGF gene expression in ALS muscle tissue.

Streamlining the polishing step development process via physicochemical characterization of monoclonal antibody aggregates

When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.

High molecular weight fibroblast growth factor 2 induces apoptosis by interacting with complement component 1 Q subcomponent-binding protein in vitro.

Fibroblast growth factor 2 (FGF2) is a multifunctional cell growth factor that regulates cell proliferation, differentiation, adhesion, migration, and apoptosis. FGF2 has multiple isoforms, including an 18‐kDa low molecular weight isoform (lo‐FGF2) and 22‐, 23‐, 24‐, and 34‐kDa high molecular weight isoforms (hi‐FGF2). Hi‐FGF2 overexpression induces chromatin compaction, which requires the mitochondria and leads to apoptosis. Complement component 1 Q subcomponent–binding protein (C1QBP) plays an important role in mitochondria‐dependent apoptosis by regulating the opening of the mitochondrial permeability transition pore. However, the interaction between C1QBP and hi‐FGF2 and its role in hi‐FGF2–mediated apoptosis remain unclear. Here, we found that hi‐FGF2 overexpression induced depolarization of the mitochondrial membrane, cytochrome c release into the cytosol, and a considerable increase in C1QBP messenger RNA and protein expression. Furthermore, coimmunoprecipitation results showed that the mitochondrial protein, C1QBP, interacts with hi‐FGF2. C1QBP knockdown using small interfering RNA significantly decreased the localization of hi‐FGF2 to the mitochondria and increased the rate of apoptosis. Our results highlight a novel mechanism underlying hi‐FGF2–induced, mitochondria‐driven cell death involving the direct interaction between hi‐FGF2 and C1QBP and the upregulation of C1QBP expression.

Gene expression levels of Casein kinase 1 (CK1) isoforms are correlated to adiponectin levels in adipose tissue and site-specific phosphorylation mediated by CK1 influences multimerization of adiponectin

Adiponectin, an adipocytokine with anti-inflammatory, antidiabetic, anti-atherogenic, and anti-cancer features, is mainly secreted by adipose tissue. Human adiponectin has an N-terminal collagenous domain and a C-terminal globular domain and builds characteristic homomeres including trimers, hexamers and high molecular weight (HMW) 12 – 18-mers. The beneficial effects of adiponectin in humans are primarily mediated by its HMW isoform. Posttranslational modifications play an important role in regulating activity and complex formation of adiponectin. Phosphorylation is considered to be another most common posttranslational modification that may alter the activity, life span, or cellular localization of proteins.

PO-445 E7107 treatment results in aberrantly spliced transcripts and protein products of P53 pathway genes

IntroductionRecent studies indicate that E7107, a spliceosome inhibitor, causes altered splicing of key genes in CLL. We evaluated the effect of E7107 on cell viability and key proteins in the p53 pathway.Material and methodsEight leukaemia/lymphoma cell lines and eight primary CLL samples (6 wild-type and 2 mutant for SF3B1) were exposed to E7107 (H3 Biomedicine) for 72 and 48 hours. Cell viability was assessed by XTT assay. To understand the effect of splicing modulation on key proteins in the p53 pathway, including p21 and MDM2, five B-cell lines were treated with E7107 for 24 hours.Results and discussionsE7107 decreased cell viability at low nanomolar concentrations in all CLL samples (mean LC50=10.5±2.0 nM; but >300 nM in two healthy PBMC controls). No correlation between drug sensitivity and SF3B1 status was observed in CLL samples (p=0.5). Six out of eight cell lines were sensitive to E7107 (mean GI50=6±1.8 nM). The GI50 values were 60.2 and 203.5 nM for the resistant HEL and HAL-01 cells, respectively. The most frequently mutated regions of the SF3B1 gene, exons 14, 15 and 16, had no mutations detected by Sanger sequencing. There was no correlation between drug sensitivity and TP53 status.Western immunoblot revealed a marked decrease of MDM2 protein level in all cell lines, accompanied by a reciprocal concentration-dependent increase in p53. The normal molecular weight p21 disappeared at higher doses of E7107, with concomitant appearance of a high molecular weight p21 isoform (~30 kDa) in TP53 wild-type cells. In addition, a lower molecular weight p53 isoform was seen in OCI-Ly3 cells treated with 50 nM E7107, likely due to alternative splicing. RT-PCR of p21 mRNA, spanning exons 3 to 4, revealed an additional longer transcript in OCI-Ly3 cells treated with E7107(50 nM, 24 hours), which sequencing confirmed was due to intron retention, predicted to result in a high molecular weight protein with an alternative c-terminal sequence. RT-PCR of MDM2 mRNA spanning exons 1 to 11 detected an altered MDM2 transcript with exons 3–10 spliced out.ConclusionE7107 treatment resulted in decreased viability of B-cell lines and primary CLL samples independently of SF3B1 status. This was associated with the production of an aberrant high molecular weight isoform of p21 due to intron retention, and a short isoform of MDM2 missing exons 3–10. Loss of normal MDM2 was accompanied by increased p53. Further investigation is needed to understand the contribution of abnormal isoforms to cell fate.

Analysis of High Molecular Weight Isoforms of Nesprin-1 and Nesprin-2 with Vertical Agarose Gel Electrophoresis.

The biochemical characterization of proteins most often require their identification by immunoblotting. Whereas SDS-PAGE provides satisfactory results for most proteins, the identification of larger proteins requires alternative methods to ensure their separation and complete transfer onto nitrocellulose membranes. Here, we describe the application of vertical agarose gel electrophoresis to identify large isoforms of nesprin-1 and nesprin-2.